Mutation Research, 283 (1992) 21-28
21
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MU'FLET 0696
5-Azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis H. Stopper, R. Pechan and D. Schiffmann Institute of Pharmacology and Toxicology, University of Wiirzburg, W-8700 Wiirzburg, Germany (Received 20 February 1992) (Revision received 28 April 1992) (Accepted 29 April 1992)
Keywords: 5-Azacytidine; Micronuclei; Kinetochores; Unscheduled DNA synthesis; Cell transformation
Summary It is known that 5-azacytidine (5-AC) induces tumors in several organs of rats and mice. The mechanisms of these effects are still poorly understood although it is known that 5-AC can be incorporated into DNA. Furthermore, it can inhibit DNA methylation. The known data on its clastogenic a n d / o r gene mutation-inducing potential are still controversial. Therefore, we have investigated the kinds of genotoxic effects caused by 5-AC in Syrian hamster embryo (SHE) fibroblasts. Three different endpoints (micronucleus formation, unscheduled D N A synthesis (UDS) and cell transformation) were assayed under similar conditions of metabolism and dose at target in this cell system. 5-AC induces morphological transformation of SHE cells, but not UDS. Therefore, 5-AC does not seem to cause repairable D N A lesions. Furthermore, our studies revealed that 5-AC is a potent inducer of micronuclei in the SHE system. Immunocytochemical analysis revealed that a certain percentage of these contain kinetochores indicating that 5-AC may induce both clastogenic events and numerical chromosome changes.
5-Azacytidine (5-AC) is an effective antineoplastic compound which has been used in the treatment of leukemia (Saiki et al., 1978). It induces tumors in several organs of rats (Carr et al., 1984) and mice in utero (Schmahl et al., 1985). 5-AC is a cytidine analog containing a nitrogen atom at the 5 position instead of a carbon. Incor-
Correspondence: Dr. D. Schiffmann, Institute of Toxicology, University of Wiirzburg, Versbacher Str. 9, W-8700 Wiirzburg, Germany.
poration of 5-AC into D N A results in the inhibition of D N A synthesis as well as D N A methylation. SOS-dependent mutagenic activity of 5-AC in Salmonella was shown by Schmuck et al. (1986). Mutagenic activity of 5-AC was observed in yeast by Zimmermann and Scheel (1984) and in Drosophila by Katz (1985). In the yeast system it also induced mitotic recombination but no chromosomal malsegregation. In mammalian cells 5-AC was not mutagenic in C 3 H / 1 0 T 1 / 2 and V79 cells at the ouabain and the hypoxanthine-guanine-phosphoribosyl-
22 transferase (hgprt) loci (Landolph and Jones, 1982). It induced chromosome decondensation, sister-chromatid exchanges, and endoreduplications (Hori, 1983). 5-AC induced mutations in the thymidine kinase (tk) +/- human lymphoblastoid line TK6 at both the tk and the hgprt locus (Call et al., 1986). Interestingly, it was 5-10 times more effective at the tk locus than at the hgprt locus. It did not induce chromosomal aberrations due to clastogenic events in TK6 cells (Call et al., 1986). Amacher and Turner (1987) found 5-AC to be mutagenic at the tk locus in L5178Y mouse lymphoma ceils. In another study (using the same cell system) by McGregor et al. (1989) 5-AC induced mutant colonies in the tk assay, but not the hgprt assay. Major perturbations of cell-cycle kinetics together with elevated rates of endomitosis and tetraploidization were found in human fibroblast-like ceils from skin and amniotic fluid (Poot et al., 1990). A block in the G 2 phase of the cell cycle was also reported by Zatsepina et al. (1989) in pig kidney cells. 5-AC transformed cultures of 10T1/2 and M2 mouse fibroblasts (Benedict et al., 1977; Harrison et al., 1983), primary rat tracheal epithelial cells (Walker and Nettesheim, 1986) and Balb/c-3T3 cells (Lubet et al., 1990). Since the data on 5-AC-induced mutagenic or clastogenic effects are still controversial, we investigated the genotoxic effects caused by this agent in Syrian hamster embryo (SHE) fibroblasts. We assayed three different biological endpoints (micronucleus formation, unscheduled DNA synthesis (UDS), and cell transformation) under similar metabolic conditions and dose at target in this cell system. Materials and methods
Chemicals
4-Nitroquinoline-l-oxide (4-NQO), hydroxyurea, Bisbenzimide 33258, 5-azacytidine and FITC-conjugated goat anti-human antibody were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was acquired from Aldrich Company Europe (Nettetal, Germany). [3H]Thymidine (spec. act. 20 Ci/mM) was obtained from Amersham Buchler Co. (Braunschweig, Germany), and CREST serum
(anti-kinetochore-antibody) was purchased from Antibodies Incorporated (Davis, CA, USA). Cell culture
SHE cells were established as described previously (Schiffmann et al., 1984). All experiments were performed with tertiary or quarternary cultures derived from 13-day-old Syrian hamster embryos. Cell cultures were grown in a humidified atmosphere with 12% CO 2 in air at 37°C. The culture medium used was IBR-modified Dulbecco's reinforced medium (Grand Island Biological Co.), supplemented with 100 U / m l penicillin and 100 /zg/ml streptomycin, 3.7 g / l NaHCO 3 and 15% fetal calf serum (Gibco, Karlsruhe, Germany). UDS assay
The UDS test was carried out according to the combined and modified procedures of Martin et al. (1978) and Lake et al. (1978). An arginine-deficient treatment in combination with short hydroxyurea treatment of the cells was applied to achieve a minimum background of replicative DNA synthesis (Schiffmann et al., 1983). Cells (1.7 x 105) were plated in triplicate on 30-mm tissue-culture Petri dishes (Falcon Plastics, Oxnard, USA) in 2 ml complete medium. After incubation for 48 h at 37°C, the medium was replaced with arginine-free medium containing 2.5% dialyzed fetal calf serum (FCS); after 24 h this medium was again replaced by fresh arginine-free medium and incubation continued for a further 48 h. At the end of this period this medium was again replaced by 1.2 ml argininefree medium containing the indicated doses of test chemicals dissolved in DMSO (final concentration 0.1%), [3H]thymidine (10 /~Ci/ml) and hydroxyurea (10 mM). All dishes were then incubated for 5 h at 37°C for DNA binding and repair. 4-NQO was used as positive control. UDS measurement was carried out according to a modified procedure previously published (Schiffmann et al., 1983). The treated cells were washed with 3 ml phosphate-buffered saline (PBS), and 1 ml of 0.1% trypsin was added to remove the cells from the dishes; solubilization of the cells was achieved by subsequent addition of 1 ml of 2% sodium dodecylsulfate. Ice-cold trichloroacetic acid (2 ml,
23 20%) was then added to each sample and the resulting precipitate was collected on 24-mm W h a t m a n G F / C glass fiber filters; the precipitate was washed 4 times with 5 ml of 5% ice-cold trichloroacetic acid and twice with absolute ethanol. The filters were then treated with 0.5 ml tissue solubilizer (Protosol, New England Nuclear, Boston, MA, USA) for several hours at 50°C and after addition of 2 0 / z l of glacial acetic acM and toluene scintillator, the radioactivity was determined in a Packard scintillation counter, model 544.
In vitro transformation assay For the transformation assay in S H E cells, a feeder layer of 2 x 10 4 lethally X-irradiated S H E cells (50 Gy) was seeded in 3 ml complete medium (IBR supplemented with 20% FCS) on 60-mm Petri dishes (Falcon Plastics, Oxnard, USA). After 24 h 150-200 target cells suspended in 1 ml medium were added and, after a further 24 h, various amounts of the test compounds dissolved in DMSO. The final concentration of D M S O in the medium did not exceed 0.1% (v/v). Following a 48-h incubation period in a humidified incubator at 12% CO 2 and 37°C, the medium was removed, the cells were washed with PBS and supplied with fresh culture medium. Eight days later, the cells were fixed with methanol, stained with 10% aqueous Giemsa and scored for cloning efficiency and morphological transformation according to the criteria (altered colony morphology consisting of criss-crossing and piling up of cells) described previously in detail by Berwald and Sachs (1965), DiPaolo et al. (1971) and Pienta (1980). Cell survival was determined in parallel by the conventional colony assay. After 10-12 days of incubation, fixation and staining, colonies with more than 50 cells were counted as survivors. In vitro micronucleus assay and kinetochore analysis S H E cells were plated in 35-mm Petri dishes containing glass coverslips and grown to medium density. Then the cell culture medium was replaced by culture medium containing 5-AC (diluted in D M S O ) or D M S O (final maximum concentration: 1% v / v ) . Following an incubation period of 5 h the compound was removed by
changing the medium. After 15 h (or different time points for time course) fixation was performed with methanol for at least 30 min at - 2 0 ° C . The cells were then stained (1 /~g/ml Bisbenzimide 33258, 5 min), washed 3 times with distilled water or PBS and mounted in Permafluor (Dianova) for microscopy. Micronuclei were determined at 1250 x magnification. Kinetochore staining was achieved by incubating the fixed cell preparations (after rinsing with PBS) with C R E S T serum (60 min) in a humidified chamber at 37°C. After rinsing with PBS again, the cells were incubated as before with FITCconjugated goat anti-human antibody (diluted 1:100 in PBS), rinsed again and counterstained with Bisbenzimide 33258 as described. Micronuclei were scored as such when up to three per cell were present. Cells with higher numbers of micronuclei were not registered. Mitotic index (cells in mitosis per 1000 cells) was determined in the same cell preparations that had been evaluated for the time course of micronucleus induction. Results
UDS assay The data obtained with 5-AC for UDS in S H E cells are given in Table 1. No UDS, i.e., no significant incorporation of [3H]thymidine exceeding that of background incorporation, was observed with 5-AC. At the highest dose tested (2.0 p.M) the level of replicative D N A synthesis was reduced to 84% of the control level indicating toxicity.
TABLE 1 UNSCHEDULED DNA SYNTHESIS (UDS) INDUCED BY 5-AC AND 4-NQO IN SHE CELLS Compound DMSO 4-NQO 5-AC
Concentration 1% 5.0 ~M 0.2/zM 0.4/zM 1.0 p.M 2.0/zM
cpm/5 x 105 cells 9870+ 683 58320_+ 1422 9853_+ 648 9829_+ 615 9141_+ 606 8323_+ 567
Each data point represents the mean of three chemically treated cultures from one experiment. The experiments were repeated three times with consistent results.
24 TABLE 2
Micronuclei/2000 Cells
MORPHOLOGICAL TRANSFORMATION CELLS, INDUCED BY 5-AC AND 4-NQO
OF
Compound
Significance (%)
DMSO 4-NQO 5-AC
Concentration
0.1% 0.01/xM 0.2/~M 0.4/.~M 1.0 p.M 2.0/,~M
Survival (%)
100 91.2 89.6 88.1 71,0 66.5
Transformation frequency Ratio
(%)
0/896 3/817 3/803 8/789 4/637 2/596
0 0.37 0.37 1.01 0.63 0.34
SHE
< 10 < 10
21
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MU'FLET 0696
5-Azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis H. Stopper, R. Pechan and D. Schiffmann Institute of Pharmacology and Toxicology, University of Wiirzburg, W-8700 Wiirzburg, Germany (Received 20 February 1992) (Revision received 28 April 1992) (Accepted 29 April 1992)
Keywords: 5-Azacytidine; Micronuclei; Kinetochores; Unscheduled DNA synthesis; Cell transformation
Summary It is known that 5-azacytidine (5-AC) induces tumors in several organs of rats and mice. The mechanisms of these effects are still poorly understood although it is known that 5-AC can be incorporated into DNA. Furthermore, it can inhibit DNA methylation. The known data on its clastogenic a n d / o r gene mutation-inducing potential are still controversial. Therefore, we have investigated the kinds of genotoxic effects caused by 5-AC in Syrian hamster embryo (SHE) fibroblasts. Three different endpoints (micronucleus formation, unscheduled D N A synthesis (UDS) and cell transformation) were assayed under similar conditions of metabolism and dose at target in this cell system. 5-AC induces morphological transformation of SHE cells, but not UDS. Therefore, 5-AC does not seem to cause repairable D N A lesions. Furthermore, our studies revealed that 5-AC is a potent inducer of micronuclei in the SHE system. Immunocytochemical analysis revealed that a certain percentage of these contain kinetochores indicating that 5-AC may induce both clastogenic events and numerical chromosome changes.
5-Azacytidine (5-AC) is an effective antineoplastic compound which has been used in the treatment of leukemia (Saiki et al., 1978). It induces tumors in several organs of rats (Carr et al., 1984) and mice in utero (Schmahl et al., 1985). 5-AC is a cytidine analog containing a nitrogen atom at the 5 position instead of a carbon. Incor-
Correspondence: Dr. D. Schiffmann, Institute of Toxicology, University of Wiirzburg, Versbacher Str. 9, W-8700 Wiirzburg, Germany.
poration of 5-AC into D N A results in the inhibition of D N A synthesis as well as D N A methylation. SOS-dependent mutagenic activity of 5-AC in Salmonella was shown by Schmuck et al. (1986). Mutagenic activity of 5-AC was observed in yeast by Zimmermann and Scheel (1984) and in Drosophila by Katz (1985). In the yeast system it also induced mitotic recombination but no chromosomal malsegregation. In mammalian cells 5-AC was not mutagenic in C 3 H / 1 0 T 1 / 2 and V79 cells at the ouabain and the hypoxanthine-guanine-phosphoribosyl-
22 transferase (hgprt) loci (Landolph and Jones, 1982). It induced chromosome decondensation, sister-chromatid exchanges, and endoreduplications (Hori, 1983). 5-AC induced mutations in the thymidine kinase (tk) +/- human lymphoblastoid line TK6 at both the tk and the hgprt locus (Call et al., 1986). Interestingly, it was 5-10 times more effective at the tk locus than at the hgprt locus. It did not induce chromosomal aberrations due to clastogenic events in TK6 cells (Call et al., 1986). Amacher and Turner (1987) found 5-AC to be mutagenic at the tk locus in L5178Y mouse lymphoma ceils. In another study (using the same cell system) by McGregor et al. (1989) 5-AC induced mutant colonies in the tk assay, but not the hgprt assay. Major perturbations of cell-cycle kinetics together with elevated rates of endomitosis and tetraploidization were found in human fibroblast-like ceils from skin and amniotic fluid (Poot et al., 1990). A block in the G 2 phase of the cell cycle was also reported by Zatsepina et al. (1989) in pig kidney cells. 5-AC transformed cultures of 10T1/2 and M2 mouse fibroblasts (Benedict et al., 1977; Harrison et al., 1983), primary rat tracheal epithelial cells (Walker and Nettesheim, 1986) and Balb/c-3T3 cells (Lubet et al., 1990). Since the data on 5-AC-induced mutagenic or clastogenic effects are still controversial, we investigated the genotoxic effects caused by this agent in Syrian hamster embryo (SHE) fibroblasts. We assayed three different biological endpoints (micronucleus formation, unscheduled DNA synthesis (UDS), and cell transformation) under similar metabolic conditions and dose at target in this cell system. Materials and methods
Chemicals
4-Nitroquinoline-l-oxide (4-NQO), hydroxyurea, Bisbenzimide 33258, 5-azacytidine and FITC-conjugated goat anti-human antibody were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was acquired from Aldrich Company Europe (Nettetal, Germany). [3H]Thymidine (spec. act. 20 Ci/mM) was obtained from Amersham Buchler Co. (Braunschweig, Germany), and CREST serum
(anti-kinetochore-antibody) was purchased from Antibodies Incorporated (Davis, CA, USA). Cell culture
SHE cells were established as described previously (Schiffmann et al., 1984). All experiments were performed with tertiary or quarternary cultures derived from 13-day-old Syrian hamster embryos. Cell cultures were grown in a humidified atmosphere with 12% CO 2 in air at 37°C. The culture medium used was IBR-modified Dulbecco's reinforced medium (Grand Island Biological Co.), supplemented with 100 U / m l penicillin and 100 /zg/ml streptomycin, 3.7 g / l NaHCO 3 and 15% fetal calf serum (Gibco, Karlsruhe, Germany). UDS assay
The UDS test was carried out according to the combined and modified procedures of Martin et al. (1978) and Lake et al. (1978). An arginine-deficient treatment in combination with short hydroxyurea treatment of the cells was applied to achieve a minimum background of replicative DNA synthesis (Schiffmann et al., 1983). Cells (1.7 x 105) were plated in triplicate on 30-mm tissue-culture Petri dishes (Falcon Plastics, Oxnard, USA) in 2 ml complete medium. After incubation for 48 h at 37°C, the medium was replaced with arginine-free medium containing 2.5% dialyzed fetal calf serum (FCS); after 24 h this medium was again replaced by fresh arginine-free medium and incubation continued for a further 48 h. At the end of this period this medium was again replaced by 1.2 ml argininefree medium containing the indicated doses of test chemicals dissolved in DMSO (final concentration 0.1%), [3H]thymidine (10 /~Ci/ml) and hydroxyurea (10 mM). All dishes were then incubated for 5 h at 37°C for DNA binding and repair. 4-NQO was used as positive control. UDS measurement was carried out according to a modified procedure previously published (Schiffmann et al., 1983). The treated cells were washed with 3 ml phosphate-buffered saline (PBS), and 1 ml of 0.1% trypsin was added to remove the cells from the dishes; solubilization of the cells was achieved by subsequent addition of 1 ml of 2% sodium dodecylsulfate. Ice-cold trichloroacetic acid (2 ml,
23 20%) was then added to each sample and the resulting precipitate was collected on 24-mm W h a t m a n G F / C glass fiber filters; the precipitate was washed 4 times with 5 ml of 5% ice-cold trichloroacetic acid and twice with absolute ethanol. The filters were then treated with 0.5 ml tissue solubilizer (Protosol, New England Nuclear, Boston, MA, USA) for several hours at 50°C and after addition of 2 0 / z l of glacial acetic acM and toluene scintillator, the radioactivity was determined in a Packard scintillation counter, model 544.
In vitro transformation assay For the transformation assay in S H E cells, a feeder layer of 2 x 10 4 lethally X-irradiated S H E cells (50 Gy) was seeded in 3 ml complete medium (IBR supplemented with 20% FCS) on 60-mm Petri dishes (Falcon Plastics, Oxnard, USA). After 24 h 150-200 target cells suspended in 1 ml medium were added and, after a further 24 h, various amounts of the test compounds dissolved in DMSO. The final concentration of D M S O in the medium did not exceed 0.1% (v/v). Following a 48-h incubation period in a humidified incubator at 12% CO 2 and 37°C, the medium was removed, the cells were washed with PBS and supplied with fresh culture medium. Eight days later, the cells were fixed with methanol, stained with 10% aqueous Giemsa and scored for cloning efficiency and morphological transformation according to the criteria (altered colony morphology consisting of criss-crossing and piling up of cells) described previously in detail by Berwald and Sachs (1965), DiPaolo et al. (1971) and Pienta (1980). Cell survival was determined in parallel by the conventional colony assay. After 10-12 days of incubation, fixation and staining, colonies with more than 50 cells were counted as survivors. In vitro micronucleus assay and kinetochore analysis S H E cells were plated in 35-mm Petri dishes containing glass coverslips and grown to medium density. Then the cell culture medium was replaced by culture medium containing 5-AC (diluted in D M S O ) or D M S O (final maximum concentration: 1% v / v ) . Following an incubation period of 5 h the compound was removed by
changing the medium. After 15 h (or different time points for time course) fixation was performed with methanol for at least 30 min at - 2 0 ° C . The cells were then stained (1 /~g/ml Bisbenzimide 33258, 5 min), washed 3 times with distilled water or PBS and mounted in Permafluor (Dianova) for microscopy. Micronuclei were determined at 1250 x magnification. Kinetochore staining was achieved by incubating the fixed cell preparations (after rinsing with PBS) with C R E S T serum (60 min) in a humidified chamber at 37°C. After rinsing with PBS again, the cells were incubated as before with FITCconjugated goat anti-human antibody (diluted 1:100 in PBS), rinsed again and counterstained with Bisbenzimide 33258 as described. Micronuclei were scored as such when up to three per cell were present. Cells with higher numbers of micronuclei were not registered. Mitotic index (cells in mitosis per 1000 cells) was determined in the same cell preparations that had been evaluated for the time course of micronucleus induction. Results
UDS assay The data obtained with 5-AC for UDS in S H E cells are given in Table 1. No UDS, i.e., no significant incorporation of [3H]thymidine exceeding that of background incorporation, was observed with 5-AC. At the highest dose tested (2.0 p.M) the level of replicative D N A synthesis was reduced to 84% of the control level indicating toxicity.
TABLE 1 UNSCHEDULED DNA SYNTHESIS (UDS) INDUCED BY 5-AC AND 4-NQO IN SHE CELLS Compound DMSO 4-NQO 5-AC
Concentration 1% 5.0 ~M 0.2/zM 0.4/zM 1.0 p.M 2.0/zM
cpm/5 x 105 cells 9870+ 683 58320_+ 1422 9853_+ 648 9829_+ 615 9141_+ 606 8323_+ 567
Each data point represents the mean of three chemically treated cultures from one experiment. The experiments were repeated three times with consistent results.
24 TABLE 2
Micronuclei/2000 Cells
MORPHOLOGICAL TRANSFORMATION CELLS, INDUCED BY 5-AC AND 4-NQO
OF
Compound
Significance (%)
DMSO 4-NQO 5-AC
Concentration
0.1% 0.01/xM 0.2/~M 0.4/.~M 1.0 p.M 2.0/,~M
Survival (%)
100 91.2 89.6 88.1 71,0 66.5
Transformation frequency Ratio
(%)
0/896 3/817 3/803 8/789 4/637 2/596
0 0.37 0.37 1.01 0.63 0.34
SHE
< 10 < 10
